Scalable purification method for aav1

ABSTRACT

A two-step chromatography purification scheme is described which selectively captures and isolates the genome-containing rAAV vector particles from the clarified, concentrated supernatant of a rAAV production cell culture. The process utilizes an affinity capture method performed at a high salt concentration followed by an anion exchange resin method performed at high pH to provide rAAV vector particles which are substantially free of rAAV intermediates.

BACKGROUND OF THE INVENTION

This invention describes a novel scalable method for producing rAAVsuitable for clinical applications.

The use of recombinant adeno-associated viruses (rAAV) for a variety ofgene therapy and vaccine approaches have been described. However, evenwith these approaches, scalable methods for purification of rAAV havebeen lacking.

Adeno-associated virus (AAV), a member of the Parvovirus family, is asmall, non-enveloped virus. AAV particles comprise an AAV capsidcomposed of 60 capsid protein subunits, VP1, VP2 and VP3, which enclosea single-stranded DNA genome of about 4.7 kilobases (kb). These VP1, VP2and VP3 proteins are present in a predicted ratio of about 1:1:10, andare arranged in an icosahedral symmetry. Individual particles packageonly one DNA molecule strand, but this may be either the plus or minusstrand. Particles containing either strand are infectious. AAV isassigned to the genus, Dependovirus, because the virus was discovered asa contaminant in purified adenovirus stocks. AAV's life cycle includes alatent phase and an infectious phase. Replication occurs by conversionof the linear single stranded DNA genome to a duplex form, andsubsequent amplification, from which progeny single strands are rescued,replicated, and packaged into capsids in the presence of helperfunctions. The properties of non-pathogenicity, broad host range ofinfectivity, including non-dividing cells, and integration make AAV anattractive delivery vehicle.

Recombinant AAV particles are produced in permissive (packaging) hostcell cultures and co-expression of helper virus AAV rep and AAV capgenes are required, for replication and packaging, the recombinantgenome into the viral particle. Genes necessary for genome replication,capsid formation and genome packaging can be expressed from transfectedplasmids, integrated into the host cell genome or introduced to the cellby recombinant viruses. Typically, cells are lysed to release rAAVparticles and maximize yield of recovered rAAV. However, the cell lysatecontains various cellular components such as host cell DNA, host cellproteins, media components, and in some instances, helper virus orhelper virus plasmid DNA, which must be separated from the rAAV vectorbefore it is suitable for in vivo use. Recent advances in rAAVproduction include the use of non-adherent cell suspension processes instirred tank bioreactors and production conditions whereby rAAV vectorsare released into the media or supernatant reducing the concentration ofhost cellular components present in the production material but stillcontaining appreciable amounts of in-process impurities. See U.S. Pat.No. 6,566,118 and PCT WO 99/11764. Therefore, rAAV particles may becollected from the media and/or cell lysate and further purified.

Certain previously described purification methods for rAAV are notscalable and/or not adaptable to good manufacturing practices,including, e.g., cesium chloride gradient centrifugation and iodixanolgradient separation. See, e.g., M. Potter et al, MolecularTherapy—Methods & Clinical Development (2014), 1: 14034, pp 1-8.

US Patent Publication No. 2005/0024467 reports that rAAV capsidserotypes such as rAAV-1, 4, 5, and 8 bind weakly to anionic resinseither as purified virus stock or in the presence of in-processproduction impurities such as host cell DNA, host cell proteins, serumalbumin, media components, and helper virus components. Purification ofthose capsid serotypes is described as involving anion-exchangechromatography in combination with other purification methods, such asiodixinol density-gradient centrifugation. See, e.g., Zolotukhin et al.,Methods 28(2):158-167 (2002) and Kaludov et al., Hum. Gene Therapy13:1235-1243 (2002); and U.S. Patent Publication No. 2004/0110266 A1.However, those methods are not readily scalable to commercial scaleprocesses.

Other examples of one- or two-step ion-exchange chromatographypurification have been reported for rAAV serotypes 1, 2, 4, 5, and 8.[Brument, N, et al. (2002). Mol Ther 6: 678-686; Okada, T, et al.(2009). Hum Gene Ther 20: 1013-1021; Kaludov, N, et al (2002). Hum GeneTher 13: 1235-1243; Zolotukhin, S, et al. (2002). Methods 28: 158-167;Davidoff, A M, et al. (2004). J Virol Methods 121: 209-215]. Morerecently, an affinity media incorporating an anti-AAV VHH ligand, asingle-domain camelid antibody derivative, was described as being usefulto purify serotypes 1, 2, 3, and 5. [Hellstrom, M, et al. (2009) GeneTher 16: 521-532]. This affinity capture method focuses on purifyingrAAV vectors from in-process production components of the cell cultureincluding helper virus, as well as helper virus proteins, cellularproteins, host cell DNA, and media components present in the rAAVproduction stock. The affinity capture method described for purifyingrAAV 1, 2, 3 and 5 particles is designed to purify rAAV from host celland helper virus contaminants, but not to separate AAV particles fromempty AAV capsids lacking packaged genomic sequences. Further, it is notclear from the literature that this separation is desirable. See, e.g.,F. Mingozzi et al, Sci Transl med. 2013 Jul. 17: 5(194), avail in PMC2014 Jul. 14, which suggests it may be desirable to include emptycapsids as decoys which can be used to overcome preexisting humoralimmunity to AAV can be overcome using capsid decoys. However, otherauthors have reported increase efficacy in rAAV1 vectors when they wereseparated from empty AAV1 capsids. See, e.g., M. Urabe et al, MolecularTherapy, 13(4):823-828 (April 2006).

There remains a need for scalable methods for separatingpharmacologically active (full) rAAV particles having the desiredtransgene packaged from rAAV capsids which lack the desired transgene.

SUMMARY OF THE INVENTION

The present invention provides a scalable method for efficientlyseparating genome-containing AAV1 vector particles (full) fromgenome-deficient rAAV1 intermediates (empty capsids). Also provided arepurified AAV1 vector particles.

In one aspect, the method for separating full AAV1 viral particles fromempty AAV1 intermediates comprises subjecting a mixture comprisingrecombinant AAV1 viral particles and AAV1 vectorintermediates/byproducts to fast performance liquid chromatography(FPLC), wherein the AAV1 viral particles and AAV1 intermediates arebound to a strong anion exchange resin equilibrated at a pH of about 9.8and subjected to a salt gradient while monitoring the eluate forultraviolet absorbance at about 260 nm and about 280 nm. The AAV1 fullcapsids are collected from a fraction which is eluted when the ratio ofA260/A280 reaches an inflection point. More particularly, the fullcapsids are collected from the eluted fraction(s) characterized byhaving a higher peak (area under the curve) at an absorbance of 260 nmas compared to the peak (area under the curve) at an absorbance of 280nm. The majority of the fractions observed for the process of theinvention have a higher amount of empty capsids (higher peak/area undercurve at A280). The absorbance peak at 260 nm being equal to orexceeding the absorbance peak at 280 nm is indicative of the fractioncontaining the full capsids.

In a further aspect, the sample loaded into the fast protein liquidchromatography (FPLC) method contains full recombinant AAV1 viralparticles and AAV1 intermediates (empty capsids) that had been purifiedfrom production system contaminants using affinity capture. In oneembodiment, the affinity capture is performed using a high performanceaffinity resin having an antibody specific for AAV.

In still another aspect, a scalable method is provided for separatingfull AAV1 viral particles from AAV1 intermediates by using an anti-AAVantibody based affinity capture resin followed by an anion exchangeresin. In one embodiment, the mixture containing the AAV1 viralparticles and AAV1 intermediates is loaded onto the affinity resin in abuffer having a high salt concentrations, e.g., about 400 nM NaCl toabout 650 mM NaCl or another salt(s) having an equivalent ionicstrength. The wash step for the affinity resin is thereafter performedat an even higher salt concentration, e.g., in the range of about 750 mMto about 1M NacCl or equivalent. In one embodiment, the AAV1 mixture ismaintained at a salt concentration of about 400 mM NaCl to about 650 mMNaCl, or equivalent prior to being applied to the anion exchange resincolumn. In a further embodiment, the rAAV1 mixture is maintained at thissalt concentration following concentration and prior to loading onto theaffinity resin.

In yet another aspect, a method for separating AAV1 viral particles fromAAV1 capsid intermediates is provided, said method comprising: (a)mixing a suspension comprising recombinant AAV1 viral particles and AAV1 vector intermediates and a Buffer A comprising 20 mM to 50 mM Bis-Trispropane (BTP) and a pH of about 9.8; (b) loading the suspension of (a)onto a strong anion exchange resin column; (c) washing the loaded anionexchange resin with Buffer 1% B which comprises a salt having the ionicstrength of 10 mM to 40 mM NaCl and BTP with a pH of about 9.8; (d)applying an increasing salt concentration gradient to the loaded andwashed anion exchange resin, wherein the salt gradient is the equivalentof 10 mM to about 40 mM NaCl; and (e) collecting rAAV1 particles fromthe eluate, where the rAAV1 particles are at least about 90% purifiedfrom AAV1 intermediates.

In a further aspect, a scalable method is provided for separatingpharmacologically active recombinant AAV1 viral particles containing DNAgenomic sequences from inert genome-deficient (empty) AAV1 vectorintermediates, said method comprising: (a) forming a loading suspensioncomprising: recombinant AAV1 viral particles and empty AAV 1 capsidwhich have been purified to remove contaminants from an AAV producercell culture in which the particles and intermediates were generated;and a Buffer A comprising 20 mM Bis-Tris propane (BTP) and a pH of about9.8; (b) loading the suspension of (a) onto a strong anion exchangeresin, said resin being in a vessel having an inlet for flow of asuspension and/or solution and an outlet permitting flow of eluate fromthe vessel; (c) washing the loaded anion exchange resin with Buffer 1% Bwhich comprises 10 mM NaCl and 20 mM BTP with a pH of about 9.8; (d)applying an increasing salt concentration gradient to the loaded andwashed anion exchange resin, wherein the salt gradient ranges from 10 mMto about 190 mM NaCl, inclusive of the endpoints, or an equivalent; and(e) collecting the rAAV particles from eluate, said rAAV particles beingpurified away from AAV1 intermediates.

In a further aspect, the affinity resin separation comprises: (i)equilibrating the affinity resin with Buffer A1 which comprises about200 mM to about 600 mM NaCl, about 20 mM Tris-Cl and a neutral pH priorto applying the material to the affinity resin; (ii) washing the loadedresin of (a) with Buffer C1 which comprises about 800 mM NaCl to about1200 mM NaCl, 20 mM Tris-Cl and a neutral pH; (iii) washing the BufferC1-washed resin of (b) with Buffer A1 to reduce salt concentration; (iv)washing the affinity resin of (c) with Buffer B which comprises about200 nM to about 600 nM NaCl, 20 mM Sodium Citrate, pH about 2.4 to about3; and (v) collecting the eluate of (iv) which comprises the full AAV1particles and the empty AAV1 capsid fraction for loading onto the anionexchange resin.

In still another aspect, vector preparations are provided that have lessthan 5% contamination with AAV intermediates (including AAV emptycapsids). In another aspect, vector preparations are provided that haveless than 2% contamination with AAV empty capsids, or less than 1%contamination with AAV empty capsids. In a further aspect, AAVcompositions are provided which are substantially free of AAV emptycapsids.

Still other advantages of the present invention will be apparent fromthe detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a chromatogram of AVB affinity-purified AAV1 vectorpreparation (1.2×10¹³GC) with a single-stranded genome run on a 0.1 mLCIMac QA column. The run was performed with 20 mM Bis-Tris-Propane (BTP)pH9.5 as the loading buffer (buffer A) and 20 mM BTP pH9.5-1M NaCl asthe column strip buffer (Buffer B). An 80 CV linear salt gradient from1-25% Buffer B was used to elute vector and the column was stripped with100% Buffer B. The flow rate was maintained at 2 mL/min throughout therun. A260 (line extending highest at peak P1), A280 (line extendingsecond highest at peak P1), programmed conductivity (line extending fromY axis and connecting at ˜11%) and actual conductivity (line extendingfrom Y axis at ˜7%) profiles are shown. Percentages of the highest peakof Absorbance (mAU) are shown on they axis. Run volume (mL) is shown assolid line beneath the x axis while buffer is indicated on the x axisabove the run volume. The major peaks (labeled P1-P3) are indicated. Thepeak fraction pool boundaries are indicated by vertical dotted lines onthe x axis and the percent recovery of loaded vector genomes in eachpeak fraction pool are indicated below the axis.

FIG. 2 shows a chromatogram of AVB affinity-purified AAV1 vectorpreparation (1.8×10¹³ GC) run on a 0.1 mL CIMac QA column. The run wasperformed with 20 mM Bis-Tris-Propane (BTP) pH10.0 as the loading buffer(buffer A) and 20 mM BTP pH10.0-1M NaCl as the column strip buffer(Buffer B). A 60 CV linear salt gradient from 1-19% Buffer B was used toelute vector and the column was stripped with 100% Buffer B. The flowrate was maintained at 2 mL/min throughout the run. A260 (line extendinghighest at peak P2), A280 (line extending second highest at peak P2 andhighest at peak P3), programmed conductivity (line extending from Y axisand connecting at ˜40 mAU) and actual conductivity (line extending fromY axis and connecting at ˜15 mAU) profiles are shown. Absorbance (mAU)are shown on the y axis. Run volume (mL) is shown as solid line beneaththe x axis, while buffer is indicated on the x axis above the runvolume. The major peaks (labeled P1-P3) are indicated. The peak fractionpool boundaries are indicated by vertical dotted lines on the x axis andthe percent recovery of loaded vector genomes in each peak fraction poolare indicated below the axis.

FIG. 3 shows a chromatogram of an AVB affinity-purified AAV1 vectorpreparation (2.64×10¹⁴ GC) run on an 8 mL CIMmultus QA column and thechromatogram is shown. The run was performed with 20 mM Bis-Tris-Propane(BTP) pH9.8 as the loading buffer (buffer A) and 20 mM BTP pH9.8-1M NaClas the column strip buffer (Buffer B). A 60 CV linear salt gradient from1-19% Buffer B was used to elute vector and the column was stripped with100% Buffer B. The column was loaded at a flow rate of 10 mL/min andeluted at 20 mL/min. A260 (line extending highest at peak P1), A280(line extending second highest at peak P1 and highest at peak P2),programmed conductivity (line extending from Y axis and connecting at˜70 mAU) and actual conductivity (line extending from Y axis andconnecting at ˜85 mAU) profiles are shown. Absorbance (mAU) is shown onthe y axis. Run volume (mL) is shown as solid line beneath the x axiswhile buffer is indicated on the x axis above the run volume. The majorpeaks eluted are indicated (labelled P1-P2). The peak fraction poolboundaries are indicated by vertical dotted lines on the x axis and thepercent recovery of loaded vector genomes in each peak fraction pool areindicated below the axis.

DETAILED DESCRIPTION OF THE INVENTION

A scalable technology for production of purified rAAV1 for use in avariety of gene transfer and/or other applications is provided.Suitably, the method purifies rAAV1 viral particles from productionculture contaminants such as helper virus, helper virus proteins,plasmids, cellular proteins and DNA, media components, serum proteins,AAV rep proteins, unassembled AAV VP1, AAVV P2 and AAV VP3 proteins, andthe like. Further, the method provided herein is particularly wellsuited for separating full rAAV1 viral particles from rAAVintermediates. In addition to being useful for AAV1 as defined herein,the method is useful for certain other AAV which share certain requiredstructural with AAV1 as described herein.

In one aspect, the method for separating full AAV1 viral particles fromempty AAV1 intermediates comprises subjecting a mixture comprisingrecombinant AAV1 viral particles and AAV 1 vector intermediates to fastperformance liquid chromatography, wherein the AAV1 viral particles andAAV1 intermediates are bound to a strong anion exchange resinequilibrated at a pH of about 9.8 and subjected to a salt gradient whilemonitoring eluate for ultraviolet absorbance at about 260 nm and about280 nm, respectively.

More particularly, the presence of AAV1 capsids having genomic sequencespackaged therein (“full”) and 260/280 absorbance ratios less than 1 ischaracteristic of AAV1 intermediates as defined herein. In general, theproduction cell culture may yield a mixture of rAAV1 “full” and rAAV1“empty” or other intermediates in which 50% or greater are intermediates(including empties), at least 60% are intermediates, or greater than 70%are intermediates. In other embodiments, more or less of the genomecopies are “empty”; as a consequence, a corresponding amount of elutedfractions are characterized by having 280 nm peaks (and correspondinglarger areas under the curve which are larger than 260 nm peaks).Fractions characterized by peaks (and corresponding larger areas underthe curve at an absorbence of about 260 nm (A260) that are higher thanthe corresponding peaks at 260 nm (A260/280 ratio is >1) are highlyenriched in full rAAV1 particles. The AAV1 full capsids are collectedfrom a fraction which is eluted when the peak for A260 crosses over andexceeds the peak for A280 (i.e., reaches an inflection point).

As used herein, “recombinant AAV1 viral particle” refers tonuclease-resistant particle (NRP) which has an AAV1 capsid, the capsidhaving packaged therein a heterologous nucleic acid molecule comprisingan expression cassette for a desired gene product. Such an expressioncassette typically contains an AAV 5′ and/or 3′ inverted terminal repeatsequence flanking a gene sequence, in which the gene sequence isoperably linked to expression control sequences. These and othersuitable elements of the expression cassette are described in moredetail below and may alternatively be referred to herein as thetransgene genomic sequences. This may also be referred to as a “full”AAV capsid. Such a rAAV viral particle is termed “pharmacologicallyactive” when it delivers the transgene to a host cell which is capableof expressing the desired gene product carried by the expressioncassette.

In many instances, rAAV particles are referred to as DNase resistant(DRP). However, in addition to this endonuclease (DNase), exonucleasesmay also be used in the purification steps described herein, to removecontaminating nucleic acids. Such nucleases may be selected to degradesingle stranded DNA and/or double-stranded DNA, and RNA. Such steps maycontain a single nuclease, or mixtures of nucleases directed todifferent targets, and may be endonucleases or exonucleases.

The term “nuclease-resistant” indicates that the AAV capsid has fullyassembled around the expression cassette which is designed to deliver atransgene to a host cell and protects these packaged genomic sequencesfrom degradation (digestion) during nuclease incubation steps designedto remove contaminating nucleic acids which may be present from theproduction process.

As used herein, “AAV1 capsid” refers to the AAV capsid having the aminoacid sequence of GenBank, accession: NP_049542 (identical to GenBankAAD27757), which is incorporated by reference herein and reproduced inSEQ ID NO:1. In addition, the methods provided herein can be used topurify other AAV having a capsid highly related to the AAV1 capsid. Forexample, AAV having sequences having about 99% identity to thereferenced amino acid sequence in NP_049542 and U.S. Pat. Nos.6,759,237, 7,105,345, 7,186,552 (i.e., less than about 1% variation fromthe referenced sequence) may be purified using the methods describedherein, provided that the integrity of the ligand-binding site for theaffinity capture purification is maintained and the change in sequencesdoes not substantially alter the pH range for the capsid for the ionexchange resin purification. Methods of generating the capsid, codingsequences therefore, and methods for production of rAAV viral vectorshave been described. See, e.g., Gao, et al, Proc. Natl. Acad. Sci.U.S.A. 100 (10), 6081-6086 (2003), U.S. Pat. Nos. 6,759,237, 7,105,345,7,186,552 and US Patent Publication 2013/0045186A1.

The term “identity” or “percent sequence identity” may be readilydetermined for amino acid sequences, over the full-length of a protein,a subunit, or a fragment thereof. Suitably, a fragment is at least about8 amino acids in length, and may be up to about 700 amino acids.Examples of suitable fragments are described herein. Generally, whenreferring to “identity”, “homology”, or “similarity” between twodifferent adeno-associated viruses, “identity”, “homology” or“similarity” is determined in reference to “aligned” sequences.“Aligned” sequences or “alignments” refer to multiple nucleic acidsequences or protein (amino acids) sequences, often containingcorrections for missing or additional bases or amino acids as comparedto a reference sequence. Alignments are performed using a variety ofpublicly or commercially available Multiple Sequence Alignment Programs.Examples of such programs include, “Clustal W”, “CAP Sequence Assembly”,“MAP”, and “MEME”, which are accessible through Web Servers on theinternet. Other sources for such programs are known to those of skill inthe art. Alternatively, Vector NTI utilities are also used. There arealso a number of algorithms known in the art that can be used to measurenucleotide sequence identity, including those contained in the programsdescribed above. As another example, polynucleotide sequences can becompared using Fasta™, a program in GCG Version 6.1. Fasta™ providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences. For instance, percentsequence identity between nucleic acid sequences can be determined usingFasta™ with its default parameters (a word size of 6 and the NOPAMfactor for the scoring matrix) as provided in GCG Version 6.1, hereinincorporated by reference. Multiple sequence alignment programs are alsoavailable for amino acid sequences, e.g., the “Clustal X”, “MAP”,“PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs.Generally, any of these programs are used at default settings, althoughone of skill in the art can alter these settings as needed.Alternatively, one of skill in the art can utilize another algorithm orcomputer program which provides at least the level of identity oralignment as that provided by the referenced algorithms and programs.See, e.g., J. D. Thomson et al, Nucl. Acids Res., “A comprehensivecomparison of multiple sequence alignments”, 27(13):2682-2690 (1999).

As used herein the term “AAV1 intermediate” or “AAV1 vectorintermediate” refers to an assembled rAAV capsid which lacks genomicsequences packaged therein. These may also be termed an “empty” capsid.Such a capsid may contain no detectable genomic sequences of anexpression cassette, or only partially packaged genomic sequences whichare insufficient to achieve expression of the gene product. These emptycapsids are non-functional to transfer the gene of interest to a hostcell.

In one aspect, a method for separating rAAV1 particles having packagedgenomic sequences from genome-deficient AAV1 intermediates is provided.This method involves subjecting a suspension comprising recombinant AAV1viral particles and AAV1 capsid intermediates to fast performance liquidchromatography, wherein the AAV1 viral particles and AAV1 intermediatesare bound to a strong anion exchange resin equilibrated at a pH of about9.8 and subjected to a salt gradient while monitoring eluate forultraviolet absorbance at about 260 and about 280. Although less optimalfor rAAV1, the pH may be as low as 9.6 or as high as 10.0. In thismethod, the AAV1 full capsids are collected from a fraction which iseluted when the ratio of A260/A280 reaches an inflection point.

Fast protein liquid chromatography (FPLC), is a form of liquidchromatography that is often used to analyze or purify mixtures ofproteins. As in other forms of chromatography, separation is possiblebecause the different components of a mixture have different affinitiesfor two materials, a moving fluid (the “mobile phase”) and a poroussolid (the stationary phase). In the present method, the mobile phase isan aqueous solution, or “buffer”. The buffer flow rate may be controlledby gravity or a pump (e.g., a positive-displacement pump) and can bekept constant or varied. Suitably, the composition of the buffer can bevaried by drawing fluids in different proportions from two or moreexternal reservoirs. The stationary phase described herein is a stronganion exchange resin, typically composed of beads. These beads may bepacked into a vessel, e.g., a cylindrical glass or plastic column, oranother suitable vessel. As provided herein, volumes of the mobile phaseare described as “column volumes”. These volumes may be extrapolated toother vessel shapes and designs.

The eluate from the anion exchange resin column or other vessel ismonitored for ultraviolet absorbance at about 260 nm and 280 nm. Asprovided herein, “full” AAV1 capsids are characterized by having a UVabsorbance of about 260 nm, whereas as “empty” capsids are characterizedby having a UV absorbance of about 280 nm. Typically, the majority ofthe eluate fractions contain empty capsids and as the salt gradientprogresses, the majority of the eluate is characterized by a curve forA280 exceeding that of A260. By monitoring UV absorbance for when theeluate is characterized by the curve for A260 crossing over the curvefor A280 (ratio of A260/A280 greater than 1), one can selectivelycollect the “full capsids” until such time as the ratio reverts toA280/A260 greater than 1.

In one embodiment, this fraction(s) selectively collected at theinversion point is characterized by having the total collected rAAVcontain at least about 90% “full capsids”, and preferably, at least 95%“full capsids”. In a further embodiment, these fractions may becharacterized by having a ratio of “intermediate” to “full” less than0.75, more preferably 0.5, preferably less than 0.3.

As used herein, an “anion exchange resin” refers to an insoluble matrixor solid support (e.g., beads) capable of having a surface ionizationover a pH range of about 1 to about 14. In one embodiment, a stronganion exchange resin is a solid support having a surface coated withquaternized polyethyleneimine. An example of such a strong anionicexchange resin is the solid support of the CIMultus QA™ column. Forexample, the anion exchange resin may be a quaternary amine ion exchangeresin. In a further embodiment, the anion exchange resin comprisestrimethylamine and a support matrix comprising poly(glycidylmethacrylate—co-ethylene dimethacrylate). However, other suitable anionexchange resins may be selected. An example of such a strong anionicexchange resin is that of the POROS HQ™ column. The resins for thecolumns listed above can be obtained from Amersham/Pharmacia(Piscataway, N.J.), PerSeptive Biosystems (Foster City, Calif.),TosoHaas (Montgomeryville, Pa.) and other suppliers.

The anion exchange material may be in the form of a monolith column or atraditional bead-based column. The ion exchange material can be in acolumn having a capacity of 0 to 0.5 mL column, 1 mL column, and morepreferably, at least an 8 mL column, a 10 mL column, a 20 mL column, a30 mL column, a 50 mL column, a 100 mL column, a 200 mL column, a 300 mLcolumn, a 400 mL column, a 500 mL column, a 600 mL column, a 700 mLcolumn, an 800 mL column, a 900 mL column, a 1000 mL (1 L) column, a2000 mL (2 L) column, a 10 L column, a 20 L column, a 30 L column, a 40L column, a 50 L column, a 60 L column, a 70 L column, an 80 L column, a90 L column, a 100 L column, a 140 L column, or a column with a capacitygreater than 140 L as well as any other column with a capacity betweenthe volumes listed above. Alternatively, another vessel type may be usedto contain the anion exchange resin solid support.

Regulation of the loading and flow rate enhances separation of the emptyand full capsids. In one embodiment, the sample loading flow rate isless than or equal to the elution flow rate. For example, the loadingflow rate may be in the range of about 10 mL/min to about 40 mL/min,about 15 mL/min to about 30 mL/min, or about 20 mL/min to about 25mL/min, about 10 mL/min, about 20 mL/min, or about 30 cm/hr to about 135cm/hr, for a 8 mL monolith column. Suitable flow rates may beextrapolated for a non-monolith column.

The specification describes salt concentrations herein with reference toNaCl for convenience. However, it will be understood that another saltof an equivalent ionic strength (e.g., KCl) may be substituted therefor,another salt having a different ionic strength, but its concentrationadjusted to an equivalent ionic strength (e.g., NH₄AC), or a combinationof salts, may be substituted. The formula for ionic strength is wellknown to those of skill in the art:

${I = {\frac{1}{2}{\sum\limits_{i = 1}^{n}{c_{i}z_{i}^{2}}}}},$

where c_(i) is the molar concentration of ion i (M, mol/L), z_(i) is thecharge number of that ion, and the sum is taken over all ions in thesolution. For a 1:1 electrolyte such as sodium chloride (NaCl),potassium chloride (KCl), formate (HCO₂ ⁻), or acetate (CH₂CO₂ ⁻) (e.g.,NH₄Ac or NaAc), the ionic strength is equal to the concentration.However, for a sulfate (SO₄ ²⁻), the ionic strength is four timeshigher. Thus, where reference is made to a specific concentration ofNaCl, or a range of concentrations, one of skill in the art cansubstitute another salt, or a mixture of suitable salts, adjusted to theappropriate concentration to provide an ionic strength equivalent tothat provided for NaCl. As used herein this this may be termed a “saltequivalent”, e.g., “NaCl or equivalent”. This will be understood toinclude both a single salt, a mixture of NaCl with other salts, or amixture of salts which do not include NaCl, but which are compatiblewith the apparatus and processes (e.g., affinity and/or anion exchangeresin processes) described herein.

The novel FPLC strategy provided herein utilizes a strong anion exchangeresin complex as described herein. The anion exchange resin binds therAAV1 empty and full capsids are bound by a charge interaction while inbuffer A (the running buffer). In one embodiment, the anion exchangeresin column in equilibrated using Buffer A which contains about 200 nMNaCl to about 700 nM NaCl, or about 400 mM NaCl to about 650 mM NaCl, orsalt equivalent. Suitable buffers may include ions contributed from avariety of sources, such as, e.g., N-methylpiperazine; piperazine;Bis-Tris; Bis-Tris propane; MES, Hepes, BTP or a phosphate bufferN-methyldiethanolamine; 1,3-diaminopropane; ethanolamine; acetic acidand the like. Such buffers are generally used at a neutral pH (e.g.,about 6.5 to about 8, preferably, about 7 to about 7.5, or about 7.5).In one embodiment, a Tris buffer component is selected. In oneembodiment, Buffer A contains about 20 mM Tris-Cl, about 400 nM NaCl orequivalent, pH 7.5.

The rAAV particles and intermediates become dissociated and returns tosolution (suspension) in buffer B (the elution buffer). Buffer B is usedto equilibrate the anion exchange resin. As provided herein, Buffer B ispreferably at a pH about 9.8 (preferably 9.8). In one embodiment, thebuffer contains about 20 mM Bis-Tris Propane (BTP) and about 10 mM NaClto about 40 nM NaCl (or salt equivalent).

A mixture containing rAAV1 empty and full particles may be suspended inabout 100% Buffer A and applied to the column (vessel). The rAAV1particles and intermediates bind to the resin while other components arecarried out in the buffer. In one embodiment, the total flow rate of thebuffer is kept constant; however, the proportion of Buffer B (the“elution” buffer) is gradually increased from 0% to 100% according to aprogrammed change in concentration (the “gradient”).

In one embodiment, at least one nuclease digestion step is performedprior to loading the mixture onto the anion exchange resin, i.e., duringthe harvest of the rAAV particles and intermediates from the productioncell culture. In a further embodiment, a second nuclease digestion step(e.g., Benzonase) is performed prior to loading the mixture onto theanion exchange resin. Suitably, this may be performed during affinitycapture. For example, an additional wash step may be incorporated intothe affinity method in which the selected nuclease(s) are pre-mixed witha buffer and used in a wash step. Suitably, the buffer is at neutral pHand a relatively low salt concentration, e.g., about 10 to about 100 mM,about 20 mM to about 80 mM, about 30 mM NaCl to about 50 mL, or about 40mM, based on the ionic strength of NaCl or a salt equivalent to any ofthe preceding ranges or amounts. In one embodiment, the flow rate forthis wash step is performed at a slower rate than the other wash stepsto allow for greater exposure of the nuclease to the loaded rAAVparticles and intermediates.

In one embodiment, the salt gradient has an ionic strength equivalent toat least about 10 mM NaCl to about 200 mM NaCl or salt equivalent. Inanother embodiment the salt gradient has an ionic strength equivalent toat least about 40 mM to about 190 mM NaCl, or about 70 nM to about 170nM NaCl. In one embodiment, the AAV1 intermediates are separated fromthe anion exchange resin when the salt gradient reaches an ionicstrength equivalent to about 50 nM NaCl or greater, or about 70 nM NaClor greater.

At different points during this process, as described herein, the boundrAAV1 particles and rAAV1 empty intermediate dissociate and appear inthe effluent. The effluent passes through two detectors which measuresalt concentration (by conductivity) and protein concentration (byabsorption of ultraviolet light at a predetermined wavelength). However,other suitable detection means may be used. As each protein is eluted itappears in the effluent as a “peak” in protein concentration and can becollected for further use.

As described herein, the fractions under the 260 nm elution peakcontaining the rAAV1 viral particles (“full”) are collected andprocessed for further use. In one embodiment, the resulting rAAV1preparation or stock contains a ratio of particles to vector genomesof 1. Optionally, the rAAV1 viral particles are placed in a suspensionhaving a pH closer to a neutral pH which will be used for long-termstorage and/or delivery to patients. Such a pH may be in the range ofabout 6.5 to about 8, or about 7 to about 7.5.

In one embodiment, the particles elute in a pH of about 9.8 and the rAAVparticles are at least about 50-90% purified from AAV1 intermediates, ora pH of 9.8 and about 90% to about 99% purified from AAV1 intermediates.A stock or preparation of rAAV1 particles (packaged genomes) is“substantially free” of AAV empty capsids when the rAAV1 particles inthe stock are at least about 75% to about 100%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, or at least 99%of the rAAV1 in the stock and “empty capsids” are less than about 1%,less than about 5%, less than about 10%, less than about 15% of therAAV1 in the stock or preparation.

In a further embodiment, the average yield of rAAV particles from loadedmaterial is at least about 70%. This may be calculated by determiningtiter (genome copies) in the mixture loaded onto the column and theamount presence in the final elutions. Further, these may be determinedbased on q-PCR analysis and/or SDS-PAGE techniques such as thosedescribed herein (see figure legends) or those which have been describedin the art.

For example, to calculate empty and full particle content, VP3 bandvolumes for a selected sample (e.g., in examples herein an iodixanolgradient-purified preparation where # of GC=# of particles) are plottedagainst GC particles loaded. The resulting linear equation (y=mx+c) isused to calculate the number of particles in the band volumes of thetest article peaks. The number of particles (pt) per 20 μL loaded isthen multiplied by 50 to give particles (pt)/mL. Pt/mL divided by GC/mLgives the ratio of particles to genome copies (pt/GC). Pt/mL−GC/mL givesempty pt/mL. Empty pt/mL divided by pt/mL and x 100 gives the percentageof empty particles.

Generally, methods for assaying for empty capsids and AAV vectorparticles with packaged genomes have been known in the art. See, e.g.,Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et al., Molec.Ther. (2003) 7:122-128. To test for denatured capsid, the methodsinclude subjecting the treated AAV stock to SDS-polyacrylamide gelelectrophoresis, consisting of any gel capable of separating the threecapsid proteins, for example, a gradient gel containing 3-8%Tris-acetate in the buffer, then running the gel until sample materialis separated, and blotting the gel onto nylon or nitrocellulosemembranes, preferably nylon. Anti-AAV capsid antibodies are then used asthe primary antibodies that bind to denatured capsid proteins,preferably an anti-AAV capsid monoclonal antibody, most preferably theB1 anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000)74:9281-9293). A secondary antibody is then used, one that binds to theprimary antibody and contains a means for detecting binding with theprimary antibody, more preferably an anti-IgG antibody containing adetection molecule covalently bound to it, most preferably a sheepanti-mouse IgG antibody covalently linked to horseradish peroxidase. Amethod for detecting binding is used to semi-quantitatively determinebinding between the primary and secondary antibodies, preferably adetection method capable of detecting radioactive isotope emissions,electromagnetic radiation, or colorimetric changes, most preferably achemiluminescence detection kit.

For example, for SDS-PAGE, samples from column fractions can be takenand heated in SDS-PAGE loading buffer containing reducing agent (e.g.,DTT), and capsid proteins were resolved on pre-cast gradientpolyacylamide gels (e.g., Novex). Silver staining may be performed usingSilverXpress (Invitrogen, CA) according to the manufacturer'sinstructions. In one embodiment, the concentration of AAV vector genomes(vg) in column fractions can be measured by quantitative real time PCR(Q-PCR). Samples are diluted and digested with DNase I (or anothersuitable nuclease) to remove exogenous DNA. After inactivation of thenuclease, the samples are further diluted and amplified using primersand a TaqMan™ fluorogenic probe specific for the DNA sequence betweenthe primers. The number of cycles required to reach a defined level offluorescence (threshold cycle, Ct) is measured for each sample on anApplied Biosystems Prsim 7700 Sequence Detection System. Plasmid DNAcontaining identical sequences to that contained in the AAV vector isemployed to generate a standard curve in the Q-PCR reaction. The cyclethreshold (Ct) values obtained from the samples are used to determinevector genome titer by normalizing it to the Ct value of the plasmidstandard curve. End-point assays based on the digital PCR can also beused.

In one aspect, an optimized q-PCR method is provided herein whichutilizes a broad spectrum serine protease, e.g., proteinase K (such asis commercially available from Qiagen). More particularly, the optimizedqPCR genome titer assay is similar to a standard assay, except thatafter the DNase I digestion, samples are diluted with proteinase Kbuffer and treated with proteinase K followed by heat inactivation.Suitably samples are diluted with proteinase K buffer in an amount equalto the sample size. The proteinase K buffer may be concentrated to 2fold or higher.

Typically, proteinase K treatment is about 0.2 mg/mL, but may be variedfrom 0.1 mg/ml, to about 1 mg/mL. The treatment step is generallyconducted at about 55° C. for about 15 minutes, but may be performed ata lower temperature (e.g., about 37° C. to about 50° C.) over a longertime period (e.g., about 20 minutes to about 30 minutes), or a highertemperature (e.g., up to about 60° C.) for a shorter time period (e.g.,about 5 to 10 minutes). Similarly, heat inactivation is generally atabout 95° C. for about 15 minutes, but the temperature may be lowered(e.g., about 70 to about 90° C.) and the time extended (e.g., about 20minutes to about 30 minutes). Samples are then diluted (e.g., 1000 fold)and subjected to TaqMan analysis as described in the standard assay.

Additionally, or alternatively, droplet digital PCR (ddPCR) may be used.For example, methods for determining single-stranded andself-complementary AAV vector genome titers by ddPCR have beendescribed. See, e.g., M. Lock et al, Hu Gene Therapy Methods, Hum GeneTher Methods. 2014 April; 25(2):115-25. doi: 1.0.1089/hgtb.2013.131.Epub 2014 Feb. 14.

In one embodiment, the mixture which is applied to the anion exchangeresin has been purified from contamination with materials present fromthe production system. Suitably, the mixture comprising the recombinantAAV1 viral particles and AAV1 intermediates contains less than about 10%contamination from non-AAV viral and cellular proteinaceous and nucleicacid materials, or less than about 5% contaminants, or less than 1%contaminating viral and cellular proteinaceous and nucleic acidmaterials. Thus, the mixture loaded onto the anion exchange resin isabout 95% to about 99% free of contaminants.

As used herein, the term “contaminants” refer to host cell, viral, andother proteinaceous materials which are present in the productionculture or are by-products thereof. This term does not include rAAVparticles or rAAV intermediates having formed AAV capsids.

In one embodiment, a two-step chromatography method is used in whichaffinity capture is utilized to separate a mixture of recombinant AAV1viral particles and AAV1 capsid intermediates from production systemcontaminants. Advantageously, this processing has been found to allowapproximately 3 times to 5 times the amount of starting material (basedon the concentration of rAAV genome copies) to be processed usingapproximately 5 to 10 less resin, as compared to certain prior artapproaches (e.g., one prior art approach utilized affinity capture afteranion exchange and another utilized multiple, sequential, ion exchangeresin columns).

This affinity capture is suitably performed using an antibody-captureaffinity resin. In one embodiment, the solid support is a cross-linked6% agarose matrix having an average particle size of about 34 μm andhaving an AAV-specific antibody. An example of one such commerciallyavailable affinity resin is AVB Sepharose™ high performance affinityresin using an AAV-specific camelid-derived single chain antibodyfragment of llama origin which is commercially available from GEHealthcare (AVB Sepharose). The manufacturer's product literatureindicates that the product binds AAV1, 2, 3 and 5, but has no referenceto AAV1. The manufacturer's literature further recommends up to a 150cm/h flow rate and a relatively low loading salt concentration. Othersuitable affinity resins may be selected or designed which contain anAAV-specific antibody, AAV1 specific antibody, or other immunoglobulinconstruct which is an AAV-specific ligand. Such solid supports may beany suitable polymeric matrix material, e.g., agarose, sepharose,sephadex, amongst others.

Suitable loading amounts may be in the range of about 2 to about 5×10¹⁵GC, or less, based on the capacity of a 30-mL column. Equivalent amountsmay be calculated for other sized columns or other vessels. At thispoint prior to anion exchange resin separation as described herein, theterm “genome copy” refers to the full particles in a mixture of bothrAAV1 full particles and rAAV1 empties/intermediates.

In one embodiment, the mixture is buffer exchanged with the columnequilibration/loading buffer. The method described herein utilizes arelatively high salt concentration for loading the column. In oneembodiment, the mixture containing the AAV1 viral particles and AAV1intermediates is loaded onto the affinity resin in a buffer having ahigh salt concentrations, e.g., about 400 nM NaCl to about 650 mM NaClor another salt(s) having an equivalent ionic strength. The wash stepfor the affinity resin is thereafter performed at an even higher saltconcentration, e.g., in the range of about 750 mM to about 1M NaCl orequivalent. In one embodiment, the AAV1 mixture is maintained at a saltconcentration of about 400 mM NaCl to about 650 mM NaCl, or equivalentprior to being applied to the anion exchange resin column. In a furtherembodiment, the rAAV1 mixture is maintained at this salt concentrationfollowing concentration and prior to loading onto the affinity resin.One example of a suitable buffer is AVB Buffer A, containing about 200nM to about 600 nM NaCl, or about 400 nM NaCl, or the ionicallyequivalent of another salt, about 10 mM to about 40 mM Tris-Cl oranother buffer, at a neutral pH. The flow rate at loading may be amanufacturer's recommended value, e.g., about 149 cm/hr. A wash stepusing AVB Buffer C is applied (1M NaCl or an equivalent salt, 20 mMsodium citrate, neutral pH), followed by a wash with AVB Buffer A, anduse of AVB Buffer B for elution. In one embodiment, AVB Buffer B isabout 200 nM to about 600 nM NaCl, or about 400 nM NaCl, or theionically equivalent of another salt, about 10 mM to about 40 mMTris-Cl, or about 20 nM Tris-Cl or another buffer. In one embodiment,this step is performed at the range recommended by the manufacturer,e.g., a low pH such as, e.g., about 2.5. In one embodiment, about 2 toabout 8, or about 5 column volumes of buffer are used for these steps.

In one embodiment, at least one nuclease digestion step is performedprior to loading the mixture onto the anion exchange resin, i.e., duringthe harvest of the rAAV particles and intermediates from the productioncell culture. In a further embodiment, a second nuclease digestion stepis performed during affinity capture. For example, an additional washstep may be incorporated into the affinity method in which the selectednuclease(s) are pre-mixed with a buffer and used in a wash step.Suitably, the buffer is at neutral pH and a relatively low saltconcentration, e.g., about 20 to about 60 mM, about 30 mM NaCl to about50 mL, or about 40 mM, based on the ionic strength of NaCl or a saltequivalent to any of the preceding ranges or amounts. In one embodiment,the flow rate for this wash step is performed at a slower rate than theother wash steps to allow for greater exposure of the nuclease to theloaded rAAV particles and intermediates.

A single nuclease, or a mixture of nucleases, may be used in this step.Such nucleases may target single stranded DNA, double-stranded DNA, orRNA. While the working examples illustrate use of a deoxyribonuclease(DNase) (e.g., Benzonase or Turbonuclease), other suitable nucleases areknown, many of which are commercially available. Thus, a suitablenuclease or a combination of nucleases, may be selected. Further, thenuclease(s) selected for this step may be the same or different from thenuclease(s) used during the processing preceding the affinity step andwhich more immediately follows harvest from the cell culture.

In one embodiment, the load for the first affinity chromatography stepis obtained following harvest and subsequent processing of cell lysatesand/or supernatant of a production cell culture. This processing mayinvolve at least one of the following processes, including, optionallysis, optional collection from supernatant (media), filtrations,clarification, concentration, and buffer exchange.

Numerous methods are known in the art for production of rAAV vectors,including transfection, stable cell line production, and infectioushybrid virus production systems which include Adenovirus-AAV hybrids,herpesvirus-AAV hybrids and baculovirus-AAV hybrids. See, e.g., G Ye, etal, Hu Gene Ther Clin Dev, 25: 212-217 (December 2014); R M Kotin, HuMol Genet, 2011, Vol. 20, Rev Issue 1, R2-R6; M. Mietzsch, et al, HumGene Therapy, 25: 212-222 (March 2014); T Virag et al, Hu Gene Therapy,20: 807-817 (August 2009); N. Clement et al, Hum Gene Therapy, 20:796-806 (August 2009); DL Thomas et al, Hum Gene Ther, 20: 861-870(August 2009). rAAV production cultures for the production of rAAV virusparticlesmay require; 1) suitable host cells, including, for example,human-derived cell lines such as HeLa, A549, or 293 cells, orinsect-derived cell lines such as SF-9, in the case of baculovirusproduction systems; 2) suitable helper virus function, provided by wildtype or mutant adenovirus (such as temperature sensitive adenovirus),herpes virus, baculovirus, or a nucleic acid construct providing helperfunctions in trans or in cis; 3) functional AAV rep genes, functionalcap genes and gene products; 4) a transgene (such as a therapeutictransgene) flanked by AAV ITR sequences; and 5) suitable media and mediacomponents to support rAAV production.

A variety of suitable cells and cell lines have been described for usein production of AAV. The cell itself may be selected from anybiological organism, including prokaryotic (e.g., bacterial) cells, andeukaryotic cells, including, insect cells, yeast cells and mammaliancells. Particularly desirable host cells are selected from among anymammalian species, including, without limitation, cells such as A549,WEHI, 3T3, 10T1/2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO,WI38, HeLa, a HEK 293 cell (which express functional adenoviral E1),Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyteand myoblast cells derived from mammals including human, monkey, mouse,rat, rabbit, and hamster. The selection of the mammalian speciesproviding the cells is not a limitation of this invention; nor is thetype of mammalian cell, i.e., fibroblast, hepatocyte, tumor cell, etc.

AAV sequences may be obtained from a variety of sources. For example, asuitable AAV sequence may be obtained as described in WO 2005/033321 orfrom known sources, e.g., the American Type Culture Collection, or avariety of academic vector core facilities. Alternatively, suitablesequences are synthetically generated using known techniques withreference to published sequences. Examples of suitable AAV sequences areprovided herein.

In addition to the expression cassette, the cell contains the sequenceswhich drive expression of an AAV capsid in the cell (cap sequences) andrep sequences of the same source as the source of the AAV ITRs found inthe expression cassette, or a cross-complementing source. The AAV capand rep sequences may be independently selected from different AAVparental sequences and be introduced into the host cell in a suitablemanner known to one in the art. While the full-length rep gene may beutilized, it has been found that smaller fragments thereof, i.e., therep78/68 and the rep52/40 are sufficient to permit replication andpackaging of the AAV.

In one embodiment, the host cell contains at least the minimumadenovirus DNA sequences necessary to express an E1a gene product, anE1b gene product, an E2a gene product, and/or an E4 ORF6 gene product.In embodiments in which the host cell carries only E1, the E2a geneproduct and/or E4 ORF6 gene product may be introduced via helper plasmidor by adenovirus co-infection. In another embodiment, the E2a geneproduct and/or E4 ORF6 may be substituted by herpesvirus helperfunctions. The host cell may contain other adenoviral genes such as VAIRNA, but these genes are not required. In one embodiment, the cell useddoes not carry any adenovirus gene other than E1, E2a and/or E4 ORF6;does not contain any other virus gene which could result in homologousrecombination of a contaminating virus during the production of rAAV;and it is capable of infection or transfection by DNA and expresses thetransfected gene (s).

One cell type useful in the methods and systems described herein is ahost cell stably transformed with the sequences encoding rep and cap,and which is transfected with the adenovirus E1, E2a, and E4ORF6 DNA anda construct carrying the expression cassette as described above. Stablerep and/or cap expressing cell lines, such as B-50 (International PatentApplication Publication No. WO 99/15685), or those described in U.S.Pat. No. 5,658,785, may also be similarly employed. Another desirablehost cell contains the minimum adenoviral DNA which is sufficient toexpress E4 ORF6. Yet other cell lines can be constructed using the novelmodified cap sequences of the invention.

The preparation of a host cell involves techniques such as assembly ofselected DNA sequences. This assembly may be accomplished utilizingconventional techniques. Such techniques include cDNA and genomiccloning, which are well known and are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y., including polymerase chain reaction, syntheticmethods, and any other suitable methods which provide the desirednucleotide sequence.

The required components for AAV production (e.g., adenovirus E1a, E1b,E2a, and/or E4ORF6 gene products, rep or a fragment(s) thereof, cap, theexpression cassette, as well as any other desired helper functions), maybe delivered to the packaging host cell separately, or in combination,in the form of any genetic element which transfer the sequences carriedthereon.

Alternatively, one or more of the components required to be cultured inthe host cell to package an expression cassette in an AAV capsid may beprovided to the host cell in trans using a suitable genetic element.

Suitable media known in the art may be used for the production of rAAVvectors. These media include, without limitation, media produced byHyclone Laboratories and JRH including Modified Eagle Medium (MEM),Dulbecco's Modified Eagle Medium (DMEM), custom formulations such asthose described in U.S. Pat. No. 6,566,118, and Sf-900 II SFM media asdescribed in U.S. Pat. No. 6,723,551, each of which is incorporatedherein by reference in its entirety, particularly with respect to custommedia formulations for use in production of recombinant AAV vectors.

rAAV production culture media may be supplemented with serum orserum-derived recombinant proteins at a level of 0.5%-20% (v/v or w/v).Alternatively, as is known in the art, rAAV vectors may be produced inserum-free conditions which may also be referred to as media with noanimal-derived products. One of ordinary skill in the art may appreciatethat commercial or custom media designed to support production of rAAVvectors may also be supplemented with one or more cell culturecomponents know in the art, including without limitation glucose,vitamins, amino acids, and or growth factors, in order to increase thetiter of rAAV in production cultures.

rAAV production cultures can be grown under a variety of conditions(over a wide temperature range, for varying lengths of time, and thelike) suitable to the particular host cell being utilized. As is knownin the art, rAAV production cultures include attachment-dependentcultures which can be cultured in suitable attachment-dependent vesselssuch as, for example, roller bottles, hollow fiber filters,microcarriers, and packed-bed or fluidized-bed bioreactors. rAAV vectorproduction cultures may also include suspension-adapted host cells suchas HeLa, 293, and SF-9 cells which can be cultured in a variety of waysincluding, for example, spinner flasks, stirred tank bioreactors, anddisposable systems such as the Wave bag system.

rAAV vector particles may be harvested from rAAV production cultures bylysis of the host cells of the production culture or by harvest of thespent media from the production culture, provided the cells are culturedunder conditions known in the art to cause release of rAAV particlesinto the media from intact cells, as described more fully in U.S. Pat.No. 6,566,118). Suitable methods of lysing cells are also known in theart and include for example multiple freeze/thaw cycles, sonication,microfluidization, and treatment with chemicals, such as detergentsand/or proteases.

At harvest, rAAV production cultures may contain one or more of thefollowing: (1) host cell proteins; (2) host cell DNA; (3) plasmid DNA;(4) helper virus; (5) helper virus proteins; (6) helper virus DNA; and(7) media components including, for example, serum proteins, aminoacids, transferrins and other low molecular weight proteins.

In some embodiments, the rAAV production culture harvest is clarified toremove host cell debris. In some embodiments, the production cultureharvest is clarified by filtration through a series of depth filtersincluding, for example, a grade DOHC Millipore Millistak+HC Pod Filter,a grade A1HC Millipore Millistak+HC Pod Filter, and a 0.2 μm FilterOpticap XL10 Millipore Express SHC Hydrophilic Membrane filter.Clarification can also be achieved by a variety of other standardtechniques known in the art, such as, centrifugation or filtrationthrough any cellulose acetate filter of 0.2 μm or greater pore sizeknown in the art. Still other suitable depth filters, e.g., in the rangeof about 0.045 μm to about 0.2 μm or other filtration techniques may beused.

Suitably, the rAAV production culture harvest is treated with anuclease, or a combination of nucleases, to digest any contaminatinghigh molecular weight nucleic acid present in the production culture.The examples herein illustrate a DNAse, e.g., Benzonase® digestionperformed under standard conditions known in the art. For example, afinal concentration of 1 unit/mL to 2.5 units/mL of Benzonase® is usedat a temperature ranging from ambient temperature to 37° C. for a periodof 30 minutes to several hours, or about 2 hours. In another example, aturbonuclease is used. However, one of skill in the art may utilizeother another suitable nuclease, or a mixture of nucleases. Examples ofother suitable nuclease is described earlier in this specification.

The mixture containing full rAAV particles and rAAV intermediates(including empty capsids) may be isolated or purified using one or moreof the following purification steps: tangential flow filtration (TFF)for concentrating the rAAV particles, heat inactivation of helper virus,rAAV capture by hydrophobic interaction chromatography, buffer exchangeby size exclusion chromatography (SEC), and/or nanofiltration. Thesesteps may be used alone, in various combinations, or in differentorders. In some embodiments, the method comprises all the steps in theorder as described below.

In some embodiments, the Benzonase®-treated mixture is concentrated viatangential flow filtration (“TFF”). Large scale concentration of virusesusing TFF ultrafiltration has been described by R. Paul et al., Hu GeneTherapy, 4:609-615 (1993). TFF concentration of the feedstream enables atechnically manageable volume of feedstream to be subjected to thechromatography steps described herein and allows for more reasonablesizing of columns without the need for lengthy recirculation times. Insome embodiments, the rAAV feedstream is concentrated between at leasttwo-fold and at least ten-fold. In some embodiments, the feedstream isconcentrated between at least ten-fold and at least twenty-fold. In someembodiments, the feedstream is concentrated between at least twenty-foldand at least fifty-fold. One of ordinary skill in the art will alsorecognize that TFF can also be used at any step in the purificationprocess where it is desirable to exchange buffers before performing thenext step in the purification process.

As used herein, the singular form of the articles “a,” “an,” and “the”includes plural references unless indicated otherwise. For example, thephrase “a virus particle” includes one or more virus particles.

As used herein, the terms “comprise”, “comprising”, “contain”,“containing”, and their variants are open claim language, i.e., arepermissive of additional elements. In contrast, the terms “consists”,“consisting”, and its variants are closed claim language, i.e.,exclusive additional elements.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.” In the context of pH values, “about” refers to a variability of±0.2 from the given value. For example, “about 9.8” encompasses to 9.6to 10.0. As to other values, unless otherwise specified “about” refersto a variability of ±10% from a given value. In certain embodiments, thevariability may be 1%, 5%, 10%, or values therebetween.

While the purification methods described herein are designedparticularly for separating full rAAV1 particles from empty rAAV1intermediates, one of skill in the art may apply these techniques toother rAAV which are closely related to AAV1 as described earlier in thespecification.

In certain embodiments, a scalable method for separating full AAV1 viralparticles from AAV1 intermediates by using an anti-AAV antibody basedaffinity capture resin followed by an anion exchange resin is provided.In one embodiment, the mixture containing the AAV1 viral particles andAAV1 intermediates is loaded onto the affinity resin in a buffer havinga high salt concentrations, e.g., about 400 nM NaCl to about 650 mM NaClor another salt(s) having an equivalent ionic strength. The wash stepfor the affinity resin is thereafter performed at an even higher saltconcentration, e.g., in the range of about 750 mM to about 1M NaCl orequivalent. In one embodiment, the AAV1 mixture is maintained at a saltconcentration of about 400 mM NaCl to about 650 mM NaCl, or equivalentprior to being applied to the anion exchange resin column. In oneembodiment, the affinity capture includes a nuclease digestion step. Ina further embodiment, the rAAV1 mixture is maintained at this saltconcentration following concentration and prior to loading onto theaffinity resin.

In a further embodiment, the affinity purified mixture containing theviral particles having packaged genomic sequences are separated fromgenome-deficient AAV1 capsid intermediates by subjecting the mixture tofast performance liquid chromatography at a pH of about 10. Moreparticularly, the AAV1 viral particles and AAV1 intermediates are boundto an anion exchange resin equilibrated at a pH of about 9.8 andsubjected to a salt gradient while monitoring eluate for ultravioletabsorbance at about 260 and about 280, wherein the AAV1 full capsids arecollected from a fraction which is eluted when the ratio of A260/A280reaches an inflection point.

In one aspect, a method for separating AAV1 viral particles from AAV1capsid intermediates is provided which involves:

-   -   (a) mixing a suspension comprising recombinant AAV1 viral        particles and AAV1 capsid intermediates and a Buffer A        comprising 20 mM to 50 mM Bis-Tris propane (BTP) and a pH of        about 9.8;    -   (b) loading the suspension of (a) onto a strong anion exchange        resin column;    -   (c) washing the loaded anion exchange resin with Buffer 1% B        which comprises a salt having the ionic strength of 10 mM to 40        mM NaCl and BTP with a pH of about 9.8;    -   (d) applying an increasing salt concentration gradient to the        loaded and washed anion exchange resin, wherein the salt        gradient is the equivalent of about about 10 mM to about 400 mM        NaCl, or about 10 mM to about 200 mM, or about 10 mM to about        190 mM; and    -   (e) collecting rAAV1 particles from elute obtained at a salt        concentration equivalent to at least 70 mM NaCl, where the rAAV1        particles are at least about 90% purified from AAV1        intermediates.

In one embodiment, the intermediates are eluted from the anion exchangeresin when the salt concentration is the equivalent of greater thanabout 50 mM NaCl. In still a further embodiment, Buffer A is furtheradmixed with NaCl to a final concentration of 1M in order to form orprepare Buffer B. In yet another embodiment, the salt gradient has anionic strength equivalent to 10 mM to about 190 mM NaCl. In still afurther embodiment, the salt gradient has an ionic strength equivalentto 20 mM to about 190 mM NaCl, or about 20 mM to about 170 mM NaCl. Theelution gradient may be from 1% buffer B to about 19% Buffer B.Optionally, the vessel containing the anion exchange resin is a monolithcolumn; loading, washing, and eluting occur in about 60 column volumes.

In still a further embodiment, a method for separating recombinant AAV1viral particles containing DNA comprising genomic sequences fromgenome-deficient (empty) AAV1 capsid intermediates is provided. Themethod involves:

-   -   (a) forming a loading suspension comprising recombinant AAV1        viral particles and empty AAV1 capsid intermediates which have        been purified to remove non-AAV materials from an AAV producer        cell culture in which the particles and intermediates were        generated; and a Buffer A comprising 20 mM Bis-Tris propane        (BTP) and a pH of about 9.8;    -   (b) loading the suspension of (a) onto a strong anion exchange        resin, said resin being in a vessel having an inlet for flow of        a suspension and/or solution and an outlet permitting flow of        eluate from the vessel;    -   (c) washing the loaded anion exchange resin with Buffer 1% B        which comprises 10 mM NaCl and 20 mM BTP with a pH of about 9.8;    -   (d) applying an increasing salt concentration gradient to the        loaded and washed anion exchange resin, wherein the salt        gradient ranges from 10 mM to about 190 mM NaCl, inclusive of        the endpoints, or an equivalent; and    -   (e) collecting the rAAV particles from eluate collected        following a salt concentration of at least about 70 mM NaCl, or        an equivalent salt or ionic strength, said rAAV particles being        purified away from intermediates.

In one embodiment, the pH is 9.8 and the rAAV particles are at leastabout 90% purified from AAV1 intermediates. In a further embodiment, theaverage yield of rAAV particles is at least about 70%.

In a further embodiment, the rAAV1 producer cell culture is selectedfrom a mammalian cell culture, a bacterial cell culture, and an insectcell culture, wherein said producer cells comprise at least (i) nucleicacid sequence encoding an AAV1 capsid operably linked to sequences whichdirect expression of the AAV1 capsid in the producer cells; (ii) anucleic acid sequence comprising AAV inverted terminal repeat sequencesand genomic transgene sequences for packaging into the AAV 1 capsid; and(iii) functional AAV rep sequences operably linked to sequences whichdirect expression thereof in the producer cells. In another embodiment,producer cells further comprise helper virus sequences required forpackaging and replication of the AAV1 into a viral particle.

In still another embodiment, the material harvested from the cellculture is applied to an affinity resin to separate contaminants fromAAV1 viral particles and empty AAV1 capsid intermediates.

In a further embodiment, the affinity resin separation comprises:

-   -   (i) equilibrating the affinity resin with Buffer A1 which        comprises about 200 mM to about 600 mM NaCl, about 20 mM Tris-Cl        and a neutral pH prior to applying the material to the affinity        resin;    -   (ii) washing the loaded resin of (a) with Buffer C1 which        comprises about 800 mM NaCl to about 1200 mM NaCl, 20 mM Tris-Cl        and a neutral pH;    -   (iii) washing the Buffer C1-washed resin of (b) with Buffer A1        to reduce salt concentration;    -   (iv) washing the affinity resin of (c) with Buffer B which        comprises about 200 nM to about 600 nM NaCl, 20 mM Sodium        Citrate, pH about 2.4 to about 3; and    -   (v) collecting the eluate of (iv) which comprises the full AAV1        particles and the empty AAV1 capsid fraction for loading onto        the anion exchange resin.

The following examples are illustrative of methods for producing AAVparticles in the supernatant of cell cultures.

Example 1

AAV vectors are produced by the triple transfection method in HEK293cells as described previously (Lock et al. 2010, Hum Gene Ther, 21(1):1259-1271). Media from ten 36-layer Hyperstack cell culture vessels isharvested and treated at 37° C. with Benzonase at 25 U/mL for 2 hfollowed by a hypertonic shock with 0.5M NaCl for 2 h. The media isfilter-clarified and concentrated 10× by tangential flow filtration(TFF) and then buffer-exchanged using the same apparatus with columnequilibration/loading buffer (AVB Buffer A). This “bulk harvestmaterial” is incubated overnight at 4° C. and then filtered with a 0.2μm PES depth filter (Sartorius).

A two-step chromatography purification scheme is described whichselectively captures and isolates the genome-containing AAV vectorparticles from the clarified, concentrated supernatant of HEK 293 cellsfive days post transfection. The load for the first chromatography stepusing AVB Sepharose High Performance affinity resin (GE), consists ofthe filter-clarified, 10×TFF-concentrated supernatant harvested from ten36-layer Hyperstack cell culture vessels that have been previouslytreated at 37° C. with 958,500 units of Benzonase for 2 h followed by ahypertonic shock with 0.5M NaCl for 2 h. Prior to loading, the bulkharvest is buffer-exchanged with the column equilibration/loading buffer(AVB Buffer A) incubated overnight at 4° C., and then filtered with a0.2 μm PES depth filter (Sartorius). The sample is applied to aflow-packed, 30-ml AVB column with a bed height of approximately 15 cmusing an AKTA Avant 150 liquid chromatography station (GE) and thefollowing method:

-   -   Flow rate: 149 cm·hr⁻¹ (5 ml/min)    -   Equilibration: 3 CV AVB Buffer A (400 mM NaCl, 20 mM Tris-Cl, pH        7.5)    -   Sample Application: approx. 4200 ml    -   Wash 1: 5 CV AVB Buffer D (1.5 mM MgCl₂, 40 mM NaCl, 20 mM        Tris-Cl, pH 7.5)        -   Premix with 150 μl (37,500 u) Benzonase Nuclease        -   Reduce the flow rate to 30 cm·hr⁻¹ (5 ml/min)    -   Wash 2: 5 CV AVB Buffer C (1M NaCl, 20 mM Tris-Cl, pH 7.5)    -   Wash 3: 3 CV AVB Buffer A    -   Elution: 2 CV AVB Buffer B (400 mM NaCl, 20 mM Sodium Citrate,        pH 2.5)    -   Re-equilibration: 3 CV Poros-9 Buffer A

A volume of 500 μl of AVB Neutralization Buffer (0.01% Pluronic F-68,0.2M Bis-Tris propane, pH 9.8) is pre-added to the elution fractiontubes and upon completion of the run, the 5-ml fractions under the main280-nm elution peak (typically three fractions) are pooled and diluted50× with AEX Buffer A-9.8 (20 mM Bis-Tris Propane pH 9.8) plus PluronicF-68 (0.001% final) in a polypropylene bottle.

Anion exchange chromatography is subsequently performed to separate thefull or DNA-carrying viral particles from the contaminating emptyparticles in the second step. Specifically, the diluted column eluatefrom the capture step is applied to a pre-equilibrated CIMmultus QA-8 mlmonolith column (BIA Separations) and the following method is run:

-   -   Flow rate: 10 ml/min    -   Equilibration: 20 CV AEX Buffer 1% B (20 mM Bis-Tris Propane pH        9.8, 10 mM NaCl)    -   Sample Application: approx. 800 ml for three diluted AVB        fractions    -   Wash 1: 10 CV AEX Buffer 1% B-9.8    -   Elution: 1-21% AEX Buffer B-9.8 (20 mM Bis-Tris Propane pH 9.8,        1M NaCl)        -   Linear gradient in 67 CV @ 20 ml/min    -   Strip: 20 CV 100% AEX Buffer B-9.8    -   Re-equilibration: 10 CV AEX Buffer 1% B-9.8

A volume of 370 μl of AEX Neutralization Buffer (0.027% Pluronic F-68,1M Bis-Tris pH 6.3) is pre-added to the elution tubes to minimizeexposure of the vector to the extreme pH after elution. Finally, the10-ml fractions under the main 260-nm elution peak (about 10) areultimately pooled and concentrated/diafiltrated with a formulationbuffer using a hollow fiber membrane.

Example 2

AAV1 vector particles containing single stranded recombinant DNA genomeswere produced by triple plasmid transfection in HEK293 cells (Lock etal. 2010, Hum Gene Ther, 21(1): 1259-1271) and the clarified productionculture supernatant was purified by affinity chromatography on AVB resin(GE). The clarified supernatant was loaded to the affinity column atneutral pH in 400 mM salt and eluted with a low pH (˜2.5) buffer. Theeluate was immediately adjusted back to neutral pH and then diluted50-fold into a Bis-Tris-propane (BTP) buffer A at pH 9.5. 1.2×10¹³vector genome copies (GC) of the eluted material was loaded onto a 0.1mL CIMac-QA anion exchange column (Bia Separations) at 2 mL/min. Thecolumn was washed in buffer A with 20 mM NaCl, eluted with a shallow(20-180 mM NaCl in buffer A, 60CV) salt gradient at the same flow-rateand then stripped with high salt Buffer B (20 mM BTP pH9.5, 1M NaCl).

A chromatogram of the CIMac-QA run is shown in FIG. 1. A major peak (P1)was observed in the elution gradient and the A260/A280 ratio of thispeak was greater than one, as would be expected for a pure particlepopulation containing vector genomes. Notably, 65% of the vector genomesloaded to the column were present in this peak as measured by thepolymerase chain reaction. A smaller peak (P2) was also observed in thegradient and in this case the peak absorbance at 280 nm is higher thanat 260 nm. A third peak (P3) was observed in the column strip and alsohad an A260/280 ratio of less than one. A low A260/280 peak ratio (<1)is indicative of empty AAV particles lacking a vector genome. Thus,three particle populations were separated on this run at pH9.5; thefirst peak (P1) is a full particle population and the last two peaks (P2and P3) contain particle populations with low vector genome content. Thefact that P3 is retained on the column and only elutes in very highsalt, while P2 elutes late in the elution gradient, suggests differentsurface charges for the two “empty” particle populations which likelyresults from different degrees of particle assembly and/or extents ofgenome packaging.

Example 3

A further aliquot (1.8×10¹³ GC) of the affinity-purified AAV1 vectorpreparation used in Example 2 was again loaded to a 0.1 mL CIMac-QAcolumn (Bia Separations) under identical conditions to those describedin Example 1, except that the pH of buffer A and buffer B was adjustedto pH10.

The chromatogram of this run is shown in FIG. 2. A major peak (P2) wasobserved in the elution gradient and as was the case in Example 2, thispeak had an A260/A280 ratio greater than one and contained the majorityof vector genomes. P3 eluted in the high salt column strip, containedvery few genome copies and had an A260/A280 ratio indicative of emptyparticles. As was expected, only a small proportion of vector genomesloaded to the column were contained in this peak. The presence of athird peak (P1) is inferred by the fact that at the leading edge of P2,the A280 trace is higher than the A260 trace although this ratio rapidlyinverts as the majority of the P2 particle population starts to elute.This rapid A260/280 ratio inversion indicates that the bulk of P1 iscontained within P2 and that unlike P2, particles in P1 have a lowvector genome content.

Overall, the data reveal that by raising the pH from 9.5 (in Example 1)to 10, a tighter column retention of all particle populations isobserved leading to less empty particle elution in the shallow saltgradient and more in the high salt column strip. In addition, thetighter retention of P2 and later elution of this peak in the saltgradient, reveals the presence of a new particle population (P1) elutingat the leading edge of P2. The high 260/280 ratio (>1) of the leadingedge of P1 suggests the hidden peak is a further packaging intermediatewith low vector genome content. The purification method is able toseparate full vector particles (P2) completely from empty particleintermediates in P3 but only partially from those in P1.

Example 4

A separate AVB affinity resin-purified AAV1 vector preparationcontaining 2.64×10″ GC was loaded onto an 8 mL CIMmultus-QA column inBis-Tris-propane (BTP) buffer A at pH9.8 and at 10 mL/min. The columnwas washed in buffer A with 20 mM NaCl, eluted with a shallow (10-190 mMNaCl, 60CV) salt gradient at 20 mL/min and then stripped with high saltBuffer B (20 mM BTP. pH9.8, 1M NaCl). Despite the increased amount ofvector loaded, a chromatographic profile similar to that describedpreviously (Example 3; FIG. 2) was obtained with a major peak (P1) inthe elution gradient containing the majority of the vector genomes (FIG.3).

A major difference to the previous scaled down run was observed in thatonly two peaks were obtained in this run and the 260/280 inversion atthe leading edge of the full particle peak (P1) was not observed. Thisdifference is likely due to the fact that the pH of the buffers waslowered from 10 to 9.8 in order to obtain better separation of P1 and P2at the end of the salt gradient. While this change obscures the presenceof the empty particle peak suggested in Example 3 at the leading edge ofP1, it will be obvious to those with skill in the art that furtherenrichment of the full particle population and reduction of empty capsidcontent might be achieved by sub-fractionation of P1 and avoidance ofthose fractions at the front portion of the peak.

Alternatively, the sample could be rerun at a higher pH and the saltgradient extended such that the front empty particle population and fullparticles are separated. Overall, the results in this exampledemonstrate the scalability of the purification method and therobustness of full particle enrichment with scale.

Sequence Listing Free Text

The following information is provided for sequences containing free textunder numeric identifier <223>.

SEQ ID NO: (containing free text) Free text under <223> 1 <223>Adeno-associated virus 1 vp1 capsid protein

All publications and references to GenBank and other sequences cited inthis specification, as well as priority applications U.S. ProvisionalPatent Application No. 62/322,083, filed Apr. 13, 2016 and USProvisional Patent Application No. 62/266,351, filed Dec. 11, 2015, areincorporated herein by reference in their entirely. While the inventionhas been described with reference to particularly preferred embodiments,it will be appreciated that modifications can be made without departingfrom the spirit of the invention.

1. A method for separating AAV1 viral particles having packaged genomicsequences from genome-deficient AAV1 capsid intermediates, said methodcomprising: subjecting a mixture comprising recombinant AAV1 viralparticles and AAV 1 capsid intermediates to fast performance liquidchromatography, wherein the AAV1 viral particles and AAV1 intermediatesare bound to an anion exchange resin equilibrated at a pH of about 9.8and subjected to a salt gradient while monitoring eluate for ultravioletabsorbance at about 260 and about 280, wherein the AAV1 full capsids arecollected from a fraction which is eluted when the ratio of A260/A280reaches an inflection point.
 2. The method according to claim 1, whereinthe inflection point is when the ratio of A260/A280 changes from lessthan 1 to greater than
 1. 3. The method according to claim 1, whereinthe salt gradient has an ionic strength equivalent to at least about 20mM to about 190 mM NaCl.
 4. The method according to claim 1, wherein theAAV1 intermediates are eluted from the anion exchange resin when thesalt gradient reaches an ionic strength equivalent to about 50 nM NaClor greater.
 5. The method according to claim 1, wherein the mixturecomprising the recombinant AAV1 viral particles and AAV1 capsidintermediates contains less than about 10% contamination from viral andcellular proteinaceous and nucleic acid materials.
 6. The methodaccording to claim 1, wherein the mixture is at least about 95% purifiedfrom viral and cellular proteinaceous and nucleic acid materials.
 7. Themethod according to claim 1, wherein the method has a sample loadingflow rate less than or equal to the elution flow rate.
 8. The methodaccording to claim 1, wherein the anion exchange resin is a strongresin.
 9. The method according to claim 1, wherein the anion exchangeresin is in a column.
 10. The method according to claim 1, wherein themixture comprising recombinant AAV1 viral particles and AAV 1 capsidintermediates had been purified from production system contaminantsusing affinity capture.
 11. The method according to claim 10, whereinthe affinity capture is performed using a sepharose high performanceaffinity resin.
 12. A method for separating AAV1 viral particles fromAAV1 capsid intermediates, said method comprising: (a) mixing asuspension comprising recombinant AAV1 viral particles and AAV 1 capsidintermediates and a Buffer A having a pH of about 9.8; (b) loading thesuspension of (a) onto a strong anion exchange resin column; (c) washingthe loaded anion exchange resin with Buffer 1% B which comprises a salthaving and a pH of about 9.8; (d) applying an increasing saltconcentration gradient to the loaded and washed anion exchange resin,wherein the salt gradient is sufficient to elute the rAAV1 particles;and (e) collecting rAAV1 particles which are at least about 90% purifiedfrom AAV1 intermediates.
 13. The method according to claim 12, whereinthe recombinant AAV1 viral particles and AAV 1 capsid of step (a) havebeen affinity purified at a high salt concentration.
 14. The methodaccording to claim 12 or 13, wherein the anion exchange resin is aquaternary amine ion exchange resin.
 15. The method according to claim14, wherein the anion exchange resin column comprises trimethylamine anda support matrix comprising poly(glycidyl methacrylate—co-ethylenedimethacrylate).
 16. The method according to claim 12, wherein Buffer Ais further admixed with NaCl to a final concentration of 1M in order toform or prepare Buffer B.
 17. The method according to claim 12, whereinthe salt gradient is from about 10 mM to about 190 mM NaCl or a saltequivalent.
 18. The method according to claim 12, wherein the elutiongradient is from 1% buffer B to about 19% Buffer B.
 19. The methodaccording to claim 12, wherein when the anion exchange resin column is amonolith column and where column loading, washing and elution occur inabout 60 column volumes.
 20. The method according to claim 12, whereinthe elution flow rate is from about 10 mL/min to about 40 mL/min. 21.The method according to claim 20, wherein the elution flow rate is about20 mL/min.
 22. A method for separating recombinant AAV1 viral particlescontaining DNA comprising pharmacologically active genomic sequencesfrom genome-deficient(empty) AAV1 capsid intermediates, said methodcomprising: (a) forming a loading suspension comprising: recombinantAAV1 viral particles and empty AAV 1 capsid intermediates which havebeen purified to remove non-AAV materials from an AAV producer cellculture in which the particles and intermediates were generated; and aBuffer A comprising 20 mM Bis-Tris propane (BTP) and a pH of about 9.8;(b) loading the suspension of (a) onto a strong anion exchange resin,said resin being in a vessel having an inlet for flow of a suspensionand/or solution and an outlet permitting flow of eluate from the vessel;(c) washing the loaded anion exchange resin with Buffer 1% B whichcomprises 10 mM NaCl and 20 mM BTP with a pH of about 9.8; (d) applyingan increasing salt concentration gradient to the loaded and washed anionexchange resin, wherein the salt gradient ranges from 10 mM to about 190mM NaCl, inclusive of the endpoints, or an equivalent; and (e)collecting the rAAV particles from eluate collected following a saltconcentration of at least about 70 mM NaCl, or an equivalent salt orionic strength, said rAAV particles being purified away from 1intermediates.
 23. The method according to claim 22, wherein the pH is9.8 and the rAAV particles are at least about 90% purified from AAV1intermediates.
 24. The method according to claim 22, wherein the averageyield of rAAV particles is at least about 40-70% as measured by GCtiter.
 25. The method according to claim 22, wherein the producer cellculture is selected from a mammalian cell culture, a bacterial cellculture, and an insect cell culture, wherein said producer cellscomprise at least (i) nucleic acid sequence encoding an AAV1 capsidoperably linked to sequences which direct expression of the AAV1 capsidin the producer cells; (ii) a nucleic acid sequence comprising AAVinverted terminal repeat sequences and genomic transgene sequences forpackaging into the AAV 1 capsid; and (iii) functional AAV rep sequencesoperably linked to sequences which direct expression thereof in theproducer cells.
 26. The method according to claim 22, wherein materialharvested from the cell culture is applied to an affinity resin toseparate contaminants from AAV1 viral particles and empty AAV1 capsidintermediates.
 27. The method according to claim 22, wherein theaffinity resin separation comprises: (i) equilibrating the affinityresin with Buffer A1 which comprises about 200 mM to about 600 mM NaCland a neutral pH prior to applying the material to the affinity resin;(ii) washing the loaded resin of (a) with Buffer C1 which comprisesabout 800 mM NaCl to about 1200 mM NaCl and a neutral pH; (iii) washingthe Buffer C1-washed resin of (b) with Buffer A1 to reduce saltconcentration; (iv) washing the affinity resin of (c) with Buffer Bwhich comprises about 200 nM to about 600 nM NaCl, 20 mM Sodium Citrate,pH about 2.4 to about 3; and (v) collecting the eluate of (iv) whichcomprises the full AAV1 particles and the empty AAV1 capsid fraction forloading onto the anion exchange resin.
 28. The method according to claim27, wherein the neutral pH is about 7.5.
 29. The method according toclaim 27, wherein in (iv), the pH is about 2.5.
 30. The method accordingto claim 27, wherein the equilibrating (i) Buffer A1 and/or Buffer B,independently have about 400 nM NaCl.